Detection of SARS‑CoV‑2 Delta, Omicron and XBB variant using colorimetric reverse‑transcription loop‑mediated isothermal amplification and specific primers

Low,Kheng Oon; Md Iqbal,Nurhezreen; Ahmad,Azrin; Johari,Nor Azfa

doi:10.3892/wasj.2025.338

Detection of SARS‑CoV‑2 Delta, Omicron and XBB variant using colorimetric reverse‑transcription loop‑mediated isothermal amplification and specific primers

Authors:
- Kheng Oon Low
- Nurhezreen Md Iqbal
- Azrin Ahmad
- Nor Azfa Johari
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Affiliations: Synthetic Biology and Genetic Engineering Division, Malaysia Genome and Vaccine Institute, National Institutes of Biotechnology Malaysia, 43000 Kajang, Selangor, Malaysia, Advanced Genomic and Bioinformatics Division, Malaysia Genome and Vaccine Institute, National Institutes of Biotechnology Malaysia, 43000 Kajang, Selangor, Malaysia, Structural Biology and Functional Omics Division, Malaysia Genome and Vaccine Institute, National Institutes of Biotechnology Malaysia, 43000 Kajang, Selangor, Malaysia
Published online on: March 27, 2025 https://doi.org/10.3892/wasj.2025.338
Article Number: 50
Copyright : © Low et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY 4.0].

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Abstract

Even with global vaccination efforts, COVID‑19 continues due to the emergent of new and more transmissible variants of severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) that evade immunity. Monitoring these variants is costly, often requiring whole‑genome sequencing. Therefore, there is a need for more cost‑effective and rapid alternatives for the identification of variant‑specific samples. Reverse‑transcription loop‑mediated isothermal amplification (RT‑LAMP) provides a more affordable and rapid method for detecting SARS‑CoV‑2 compared to other molecular tests. The present study designed colorimetric RT‑LAMP assays that target mutations associated with specific SARS‑CoV‑2 lineages. These assays were tested on 347 confirmed SARS‑CoV‑2 RNA samples. The results revealed promising accuracy: 92.18% for Omicron‑specific primers, followed by 80.64% for XBB and 71.57% for Delta. This assay effectively distinguishes the variants of concern, Delta, Omicron and XBB, without the use of costly equipment/facilities and does not require the specialized training of personnel. This development supports the use of RT‑LAMP as a valuable tool in healthcare systems battling COVID‑19.

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1	Konings F, Perkins MD, Kuhn JH, Pallen MJ, Alm EJ, Archer BN, Barakat A, Bedford T, Bhiman JN, Caly L, et al: SARS-CoV-2 Variants of Interest and Concern naming scheme conducive for global discourse. Nat Microbiol. 6:821–823. 2021.PubMed/NCBI View Article : Google Scholar
2	Saito A, Irie T, Suzuki R, Maemura T, Nasser H, Uriu K, Kosugi Y, Shirakawa K, Sadamasu K, Kimura I, et al: Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation. Nature. 602:300–306. 2022.PubMed/NCBI View Article : Google Scholar
3	Vega-Magaña N, Sánchez-Sánchez R, Hernández-Bello J, Venancio-Landeros AA, Peña-Rodríguez M, Vega-Zepeda RA, Galindo-Ornelas B, Díaz-Sánchez M, García-Chagollán M, Macedo-Ojeda G, et al: RT-qPCR assays for rapid detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations: A screening strategy to identify variants with clinical impact. Front Cell Infect Microbiol. 11(672562)2021.PubMed/NCBI View Article : Google Scholar
4	Sagar V, Singh MP, Kaur G, Khurana R, Agarwal R, Ratho RK, Ghosh A, Kulashri A and Aggarwal AK: LAMP-Based Point-of-Care nucleic Acid-based detection method can be useful for quick Decision-making for diagnosis of acute COVID-19 Emergency cases in Hospital Settings. COVID. 3:914–923. 2023.
5	Song X, Coulter FJ, Yang M, Smith JL, Tafesse FG, Messer WB and Reif JH: A lyophilized colorimetric RT-LAMP test kit for rapid, low-cost, at-home molecular testing of SARS-CoV-2 and other pathogens. Sci Rep. 12(7043)2022.PubMed/NCBI View Article : Google Scholar
6	Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N and Hase T: Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28(E63)2000.PubMed/NCBI View Article : Google Scholar
7	Nagamine K, Hase T and Notomi T: Accelerated reaction by Loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 16:223–229. 2002.PubMed/NCBI View Article : Google Scholar
8	Dos Santos CA, Silva LDC, Souza Júnior MN, Mendes GM, Estrela PFN, de Oliveira KG, de Curcio JS, Resende PC, Siqueira MM, Pauvolid-Corrêa A, et al: Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers. Sci Rep. 12(11500)2022.PubMed/NCBI View Article : Google Scholar
9	Chan WS, Lam YM, Law JHY, Chan TL, Ma ESK and Tang BSF: Geographical prevalence of SARS-CoV-2 variants, August 2020 to July 2021. Sci Rep. 12(4704)2022.PubMed/NCBI View Article : Google Scholar
10	Chen Z, Azman AS, Chen X, Zou J, Tian Y, Sun R, Xu X, Wu Y, Lu W, Ge S, et al: Global landscape of SARS-CoV-2 genomic surveillance and data sharing. Nat Genet. 54:499–507. 2022.PubMed/NCBI View Article : Google Scholar
11	Luo Z, Ye C, Xiao H, Yin J, Liang Y, Ruan Z, Luo D, Gao D, Tan Q, Li Y, et al: Optimization of loop-mediated isothermal amplification (LAMP) assay for robust visualization in SARS-CoV-2 and emerging variants diagnosis. Chem Eng Sci. 251(117430)2022.PubMed/NCBI View Article : Google Scholar
12	Alves PA, de Oliveira EG, Franco-Luiz APM, Almeida LT, Gonçalves AB, Borges IA, Rocha FS, Rocha RP, Bezerra MF, Miranda P, et al: Optimization and clinical validation of colorimetric reverse transcription loop-mediated isothermal amplification, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to Detect SARS-CoV-2 variants of concern. Front Microbiol. 12(713713)2021.PubMed/NCBI View Article : Google Scholar
13	Sherrill-Mix S, Van Duyne GD and Bushman FD: Molecular beacons allow specific RT-LAMP detection of B.1.1.7 variant SARS-CoV-2. J Biomol Tech. 32:98–101. 2021.PubMed/NCBI View Article : Google Scholar
14	Yang J, Hu X, Wang W, Yang Y, Zhang X, Fang W, Zhang L, Li S and Gu B: RT-LAMP assay for rapid detection of the R203M mutation in SARS-CoV-2 Delta variant. Emerg Microbes Infect. 11:978–987. 2022.PubMed/NCBI View Article : Google Scholar
15	Alhamid G, Tombuloglu H and Al-Suhaimi E: Development of loop-mediated isothermal amplification (LAMP) assays using five primers reduces the false-positive rate in COVID-19 diagnosis. Sci Rep. 13(5066)2023.PubMed/NCBI View Article : Google Scholar
16	Nawab M, Riaz SK, Ismail E, Ahamed A, Tariq A, Malik MFA, Qusty NF, Bantun F, Slama P, Umair M, et al: Integrated approach for detection of SARS-CoV-2 and its variant by utilizing LAMP and ARMS-PCR. Ann Clin Microbiol Antimicrob. 23(11)2024.PubMed/NCBI View Article : Google Scholar